IAWA Journal, Vol. 29 (3), 2008: 277–289

COMPARATIVE DEVELOPMENT OF INTERTRACHEARY PIT MEMBRANES IN ABIES FIRMA AND METASEQUOIA GLYPTOSTROBOIDES

Roland Dute, LaToya Hagler and Adam Black Department of Biological Sciences, Auburn University, Life Sciences Building, Auburn, Alabama 36849-5407, U.S.A.

SUMMARY This study compares intertracheary pit membrane structure and ontogeny in Abies firma (Pinaceae) and Metasequoia glyptostroboides (Cupres- saceae). Initial phases of pit membrane development are the same for both species. Branched plasmodesmata are present in the earliest stages of pit membrane development observed. Torus thickening of the pit membrane occurs early in pit development prior to pit border initiation. During pit border enlargement, frequently occlude the apertures. Cell lysis is associated with complete wall matrix removal from pit membranes of Metasequoia. By contrast, cell lysis in Abies results in loss of matrix ma- terial from the margo, whereas the torus remains largely unaffected. Torus extensions in pit membranes of A. firmaretain variable amounts of matrix material. Either a difference in chemical composition of the torus or a difference in autolytic enzymes is hypothesized to explain developmental differences between pit membranes of the two species. Key words: Abies, Metasequoia, pit membrane, plasmodesma, , torus, torus extension.

INTRODUCTION

Recent physiological work has shown the importance of torus-margo pitting to efficient water flow in tracheids of conifers (Hacke et al. 2004; Pittermann et al. 2005). At the same time the ability of the impermeable torus to prevent air seeding is well known (Zim- mermann 1983; Hacke et al. 2004). Earlier studies of mature pit membranes in gymno- sperms have shown the diversity of torus structure (Bauch et al. 1972). In particular, the amount of matrix material remaining within a mature torus can range from considerable (e.g. Abies – Parham & Baird 1973; Sano et al. 1999) to none (Ginkgo – Dute 1994). Metasequoia is another genus in which matrix loss from the torus occurs during cell maturation (personal observation). It was decided to compare pit membrane develop- ment in Abies firma (Pinaceae) and Metasequoia glyptostroboides (Cupressaceae). One question involves time of torus initiation. Torus development in conifers is thought to occur during the primary wall stage of growth (Bauch et al. 1968). In agreement with these findings, Parham and Baird (1973) found torus ontogeny in Abies balsamea largely completed by the beginning of pit border formation. However, Timell (1979) indicates that for this same species torus formation begins at the same time as secondary wall deposition. No information exists for time of torus initiation

Downloaded from Brill.com10/06/2021 11:20:42PM via free access 278 IAWA Journal, Vol. 29 (3), 2008 Dute, Hagler & Black — Pit membranes of Abies and Metasequoia 279 in Metasequoia. In eudicotyledons, time of initiation correlates with method of torus manufacture as well as with torus chemistry (Coleman et al. 2004). A second set of questions involves tori and plasmodesmata. Plasmodesmata are com- mon in intertracheary pit membranes of conifers and Ginkgo (q.v. discussion in Dute 1994). Various authors have shown that tori of Abies have plasmodesmata (Timell 1979; Sano et al. 1999). Although no such work has been done on Metasequoia, in Ginkgo, whose torus also loses its matrix material, plasmodesmata disappear at maturity (Dute 1994). Therefore, in the present study, particular attention was paid to plasmodesmata – their time of formation, structure, and final disposition. Finally, torus extensions are well-known from the pit membranes of various species of Abies (Bauch et al. 1972; Imamura et al. 1974; Sano et al. 1999, and literature cited therein). We decided to characterize the anatomy of these structures in Abies firma using SEM and TEM.

MATERIALS AND METHODS

Branch specimens of 2–4 mm diameter were collected from two individuals each of Abies firma Siebold et Zucc. and Metasequoia glyptostroboides Miki ex Hu & Cheng growing in the Donald E. Davis Arboretum on the Auburn University campus. Five collections were made during the spring of 2005; two collections were made during the spring of 2006. Prior investigations of macerated showed average length of branch tracheids to be 1047 μm for Metasequoia and 931 μm for Abies (based on 25 counts each). In order to minimize physical damage to immature tracheids during col- lection and preservation, the following procedure was adopted. Branch segments of approximately 6 mm length were cut and split in half. The periderm was gently scraped away. All cutting and scraping were done in fixative. The specimens were preserved in one-half strength Karnovskyʼs fixative (Karnovsky 1965) in 0.1 M sodium phosphate buffer (pH 7.0) under vacuum at room temperature for one hour. Afterward, the specimen strips were cut to lengths of 2–2.5 mm by removing tissue from both ends, and were cut longitudinally a number of times to make wedges. The wedges were placed in fresh fixative and evacuated for a further hour. The specimens then were kept at 4 °C for five hours. After repeated buffer washes, the specimens were placed in 1% buffered OsO4 for 4.5 hours at room temperature. Another series of buffer washes followed. Specimens then were dehydrated in a cold ethanol series followed by infiltration with acetone. Dehydration was succeeded by infiltration in increasing concentrations of Spurrʼs resin (Spurr 1969) in acetone. After 24 hours in pure resin the tissue was embedded in flat molds at 63 °C for 24 hours. Embedded material was mounted and sectioned for light and transmission electron microscopy (TEM) using a Sorvall MT-2b ultramicrotome. For light microscopy, 2 μm thick sections were heat-fixed to glass slides and stained with 0.5% buffered toluidine blue O. The sectioned material was viewed and photographed with a Nikon Biophot and an attached Nikon D70 camera. For TEM, sections of about 80 nm thickness were cut, placed on copper grids, and stained with uranyl acetate and lead citrate. The sec- tions were viewed with a Zeiss EM10CR using 60 kV accelerating voltage.

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For scanning electron microscopy (SEM) branches were split radially and either air- dried or preserved in FAA (formalin-acetic acid-alcohol). The latter specimens were then dehydrated through absolute ethanol, soaked in two changes of HMDS (hexamethyl- disilazane) (Nation 1983) overnight, and allowed to air dry. All dried specimens were mounted on aluminum stubs using carbon-impregnated tape and coated with gold-palla- dium using an Electron Microscopy Sciences 550X sputter coating device. The resulting preparations were viewed with a Zeiss DSM 940 at 15 kV accelerating voltage. Observations from light, SEM, and TEM were predominately from the earlywood.

RESULTS Torus initiation and ontogeny With the light microscope, well-developed tori associated with developing pit borders are frequently encountered in immature xylem of both Metasequoia and Abies (Fig. 1 & 2), indicating that tori are initiated early in cell ontogeny. In fact, exceptional views

Abbreviations used in this study: D = dictyosomes; E = ; M = mitochon- drion; MA = margo; P = plastid; PH = secondary ; PL = plasmodesma; T = torus; TE = torus extension; V = vesicles; VC = vascular cambium; W = warts; X = secondary xylem.

Figures 1–4. Initiation of torus. – 1: Light micrograph (LM) of active cambial region in Metase- quoia. – 2: LM of active cambial region in Abies. – 3: TEM of newly initiated torus thickening in Metasequoia. – 4: TEM of newly initiated torus thickening in Abies. Note branched plasmodes- mata. Unlabeled arrows indicate the beginnings of a pit border. — Scale bars = 20 μm for Fig. 1 & 2; 1 μm for Fig. 3 & 4.

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Figures 5–7. Cytoplasmic organelles associated with young pit membranes. – 5: Metasequoia. – 6: Metase- quoia. – 7: Abies. Unlabeled arrows in Fig. 5 & 6 de- note invaginations enveloping wall mate- rial. — Scale bars = 1 μm for all.

using TEM indicate that tori are initiated and can show considerable thickening by the time pit border initiation occurs (Fig. 3 & 4). Figure 4 is particularly instructive. One cell (A) is slightly older than its neighbor (B), and the former shows not only a thicker torus pad, but also initiation of the pit border (unlabeled arrows). Before and during pit border construction the torus is spatially associated with a number of cytoplasmic components, including: plastids, mitochondria, rough ER, dictyosomes, and vesicles (Fig. 5–7). More will be said of plastids later. A plexus is not associated with torus thickening.

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Figures 8–12. Matrix removal from pit mem- branes of Metasequoia. – 8: LM showing pit membrane pre- (A) and postautolysis (B). – 9: TEM of postautolysis pit membrane (sec- tional view). – 10: TEM of torus in the act of losing its matrix material (arrows). – 11: LM of pit membrane in which some matrix material (arrow) still remains in the torus. – 12: SEM of unaspirated pit membrane in sur- face view. — Scale bar = 20 μm for Fig. 8; 1 μm for Fig. 9–11; 2.5 μm for Fig. 12.

Cell autolysis follows completion of the pit border. In Metasequoia, the entire pit membrane, including the torus, loses its matrix material (Fig. 8 & 9). With TEM, both torus and margo are distinct, but their thicknesses have decreased from the pre-autolysis stage (Fig. 9). It is possible to find instances in which matrix removal is occurring. Figure 10 represents an example of the torus at this ontogenetic stage. Loss of ma- trix occurs not only from the surface of the torus, but also from sites throughout its

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Figure 13 & 14. Postautolysis pit membranes of Abies. – 13: TEM of sectional view. – 14: SEM of surface view. — Scale bar = 1 μm for Fig. 13; 2.5 μm for Fig. 14.

thickness. Figure 11 shows a situation in which matrix removal is complete in the margo while removal is still occur- ring in the torus. This situation is due probably to the greater thickness of the torus. Removal of matrix from entire pit membranes of Metasequoia allows one to visualise clearly the fibrillar compo- sition of the torus using SEM (Fig. 12).

Figure 15 & 16. Plasmodesmata in pit mem- branes of Metasequoia (Fig. 15) and Abies (Fig. 16) in sectional view. – 15: Branched plasmodesmata are present prior to torus ini- tiation. The plasmodesmata of Fig. 15 are at the periphery of the future pit membrane, at the site of what will become the margo. – 16: Detail of branched, secondary plasmo- desmata in torus thickenings. Unlabeled ar- rows indicate cell membrane invaginations containing wall components. – Scale bar = 0.5 μm for both.

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Abies, in contrast to Metasequoia, retains the matrix of its torus, although the post- autolysis torus is more electron opaque, indicating that some chemical changes have taken place (Fig. 13). Observations of the torus using SEM show either a surface with scattered microfibrils or one with “circular oriented microfibrils” as first observed for Abies by Parham et al. (1973) (Fig. 14). In both instances, matrix material is clearly present.

Plasmodesmata Complex plasmodesmata are present from the earliest stages of torus development in both Metasequoia and Abies (Fig. 4 & 6). In fact, plasmodesmata exist in the radial walls of immature tracheids even before the initiation of torus thickenings. It is unknown if these plasmodesmatal clusters represent future torus sites because these channels can also be found on the primordial pit membrane beyond the region of the future

Figures 17–20. Plasmodesmata in pit membranes of Abies in surface view. – 17: TEM of torus containing plasmodesmata. –18: SEM of aspirated pit membrane. Plasmodesmata still evident in torus. – 19, 20: Mature, unaspirated pit membranes (SEM) with irregular clusters of plasmodes- mata in tori. — Scale bar = 0.5 μm for Fig. 17; 5 μm for Fig. 18; 2.5 μm for Fig. 19 & 20.

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Figures 21–23. – 21: Mature pit membrane of Abies possessing a torus extension with empty plasmodesmatal channels. – 22: Immature sieve area between sieve cells of Metase- quoia. Note the secondary plasmodesmata. – 23: Detail (SEM) of surface of torus exten- sion in unaspirated, mature pit membrane of Abies. — Scale bar = 1 μm for Fig. 21 & 23; 0.5 μm for Fig. 22.

Figure 24 & 25. SEM of mature, aspirated pit membranes of Abies. Both torus rim and torus ex- tension are tightly pressed to the surface of the pit border. – Scale bar = 5 μm for Fig. 24; 1 μm for Fig. 25.

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torus (Fig. 15). Structurally, plasmodesmata represent branches (half plasmodesmata) extending from median channels or cavities (Fig. 16). Ultrathin sectional views often show pit membranes with few or no plasmodesmatal branches. Paradermal views of tori in mature pits of Abies indicate that although some tori do lack plasmodesmata, often the channels show an uneven distribution (Fig. 17–20). This observation would explain the apparent absence of plasmodesmata in many ultrathin cross sections as noted above. Torus development is associated with invaginations of the cell membrane contain- ing wall material (Fig. 5, 6 & 16 at arrows). Some of these invaginations appear to be associated with plasmodesmatal channels that extend from the surface of the wall into the cytoplasm. Plasmodesmatal channels are retained by the torus in Abies as it matures although the internal membranes are frequently but not always lost during autolysis. The channels are evident in tori of both aspirated and non-aspirated pit membranes seen with SEM (Fig. 18–20) as well as in thin sections (Fig. 13 & 21). The presence of median cavities does not affect the structural integrity of the torus during aspiration (Fig. 18–20). In mature pit membranes of Metasequoia, no plasmodesmata are evident (Fig. 12).

Figure 26 & 27. Association of plastids and the apertures of immature pits. – 26: Transverse section, Abies. – 27: Oblique longitudinal sec- tion, Metasequoia. — Scale bar = 1 μm for Fig. 26; 5 μm for Fig. 27.

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Median cavities with plasmodesmatal branches are not restricted to pit membranes between tracheids in Abies and Metasequoia, but are found connecting other cell types as well. The best developed examples exist in the sieve areas connecting sieve cells (Fig. 22).

Torus extensions Mature pit membranes of Abies frequently possess “torus extensions” (Imamura et al. 1974) from which margo material has not been removed or only incompletely so (Fig. 19–21). Figure 23 is a detailed view of one such extension showing distinct granules that probably represent matrix material that would have been removed had the process of hydrolysis gone to completion. The torus extensions do not inhibit the process of aspiration, at least not in the pit membranes of the earlywood (Fig. 18, 24 & 25). The torus of such air-dried pit membranes fits snugly against the aperture as indicated by a complete outline of the aperture beneath the torus. The torus extension, in such instances, is tightly appressed to the surface of the pit border such that warts of the pit border wall are visible through material of the extension.

Plastids During pit border development in both Metasequoia and Abies, the pit apertures frequently are occluded by plastids (Fig. 7 & 26). These plastid “plugs” consist of one to several plastid profiles when viewed in transverse section, but paradermal sections (parallel to the surface of the torus) indicate that only one plastid is associated with an aperture. Figure 27 provides an oblique longitudinal section of three pit pairs. Each of the three visible apertures contains a plastid.

DISCUSSION

As is true for Ginkgo, plastids often occlude apertures of developing pit borders. Timell (1979) in his study of compression wood of Abies balsamea notes in his Figure 14 that a large amyloplastid is located in a pit aperture during deposition of the S1 layer. A similar observation is made for his Figure 15, and his Figure 4 has an aperture with a plastid plug that goes unmentioned. These observations, plus those in the present paper, as well as those made by Dute (1994) for Ginkgo, would indicate a developmental phase in pit border construction during which plastids are likely to block the apertures. The metabolic importance of this situation is unknown. Initiation of torus thickening in both Abies firma and Metasequoia glyptostroboides occurs before initiation of the pit border. A similar situation is true for the tori of Taxo- dium distichum (Thomas 1972), Pinus (Imamura & Harada 1973), and Ginkgo (Dute 1994). These observations indicate that torus thickenings in consist of primary wall material. Tori of species of Ulmus and Celtis not only develop prior to pit border formation, but also are not associated with a microtubule plexus (Dute & Rushing 1990). The same correlation appears to be true of gymnosperm tori. Although plasmodesmata are not typically observed in developing and/or mature intervascular pit membranes of eudicotyledons (Barnett 1982; Dute & Rushing 1988,

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1990; Dute et al. 1990), they are always noted in pits of gymnoperms (Murmanis & Sachs 1969; Thomas 1969, 1972; Fengel 1972; Fujikawa & Ishida 1972; Timell 1979; Dute 1994). Thus, the presence of plasmodesmata in the pit membranes of Abies and Metasequoia is not surprising. The structure of the plasmodesmata in these two genera is identical to that of the other gymnosperms so far investigated. These cytoplasmic channels are referred to as “secondary plasmodesmata” in the literature and consist of multiple cytoplasmic channels emanating from median cavities (Evert 2006). The radial walls studied are non-division walls; thus, plasmodesmata at these sites should be secondary (Evert 2006). According to Kollmann and Glockmann (1991), the initial phase of secondary plasmodesmata formation is a “localized thinning of cell walls” in preparation for fusion of the plasmalemmas and ER strands of adjacent cells. Therefore, in Abies and Metasequoia one would expect initiation of secondary plasmodesmata to occur prior to wall thickening associated with torus formation. With the advent of such thickening, the already established plasmodesmatal channels would merely increase in length. Removal of matrix leads to a realignment of cellulose microfi- brils (McCann et al. 1990). Such an occurrence would explain the disappearance of plasmodesmata from mature tori of Metasequoia but their retention in mature tori of Abies. The function of plasmodesmata in the intercellular trafficking of is well- known (Kragler et al. 1998). The specific molecular traffic between immature tracheids is, however, unknown. One possibility could be growth hormones. Kwiatkowska (1991) found that uptake of gibberellic acid into antheridial cells of Chara vulgaris was cor- related with the presence of structurally intact plasmodesmata. It is unknown why matrix removal occurs from tori of M. glyptostroboides but not from tori of A. firma. Two possibilities suggest themselves: 1) a difference in chemical composition of the tori or 2) a difference in autolytic enzymes. It should be possible to test torus composition in immature tracheids using TEM and confocal microscopy (Morrow & Dute 1999; Coleman et al. 2004). Torus extensions have been described for species of Abies, Juniperus, and Tsuga (q.v. discussion in Sano et al. 1999). Both Liese (1965) and Sano et al. (1999) suggest that the torus extensions and the torus itself are identical chemically. However, in Abies firma the torus extensions are removed by hemicellulase, whereas the torus is degraded by pectinase (Imamura et al. 1974). Banks (1971) pictures a partially aspirated pit membrane in air-dried A. grandis earlywood. In contrast, we show in the present manuscript that in the earlywood (where most of the water conduction would be expected to occur), the torus extensions do not interfere with aspiration. In Krahmer & Côté (1963) no mention was made of torus extensions impeding aspiration in two species of Tsuga. Sano et al. (1999) have shown for A. sachalinensis that the frequency of pit membranes with extended tori, and the areal extent of the margo occupied by the extensions within individual pit membranes increase from early- to latewood. Fujikawa and Ishida (1972) provided evidence that latewood pit membranes of the “rich amorphous type” (in which the margo was “in- crusted with amorphous substances”) found in some conifers did not aspirate upon

Downloaded from Brill.com10/06/2021 11:20:42PM via free access 288 IAWA Journal, Vol. 29 (3), 2008 Dute, Hagler & Black — Pit membranes of Abies and Metasequoia 289 air-drying. Although the authors investigated three species of Abies, no such claim was made for the latewood pit membranes of this genus. In summary, the synthetic pathways of pit membrane development in Abies firmaand in Metasequoia glyptostroboides are similar to one another and to other gymnosperms thus far studied. This similarity includes a stage during which the developing apertures are associated with plastid plugs. However, pit membranes of Abies and Metasequoia respond differently to autolysis leading to differential amounts of matrix removal.

REFERENCES

Banks, W.B. 1971. Structure of the bordered pit membrane in certain softwoods as seen by scanning electron microscopy. J. Inst. Wood Sci. 5 (4): 12–15. Barnett, J.R. 1982. Plasmodesmata and pit development in secondary xylem elements. Planta 155: 251–260. Bauch, J., W. Liese & F. Scholz. 1968. Über die Entwicklung und stoffliche Zusammensetzung der Hoftüpfelmembranen von Längstracheiden in Coniferen. Holzforschung 22: 144–153. Bauch, J., W. Liese & R. Schultze. 1972. The morphological variability of the bordered pit mem- branes in gymnosperms. Wood Sci. Technol. 6: 165–184. Coleman, C.M., B.L. Prather, M.J. Valente, R.R. Dute & M.E. Miller. 2004. Torus lignification in hardwoods. IAWA Bull. n.s. 25: 435–447. Dute, R.R. 1994. Pit membrane structure and development in Ginkgo biloba. IAWA J. 15: 75–90. Dute, R.R. & A.E. Rushing. 1988. Notes on torus development in the wood of Osmanthus americanus (L.) Benth. & Hook ex Gray (Oleaceae). IAWA Bull. n.s. 9: 41–51. Dute, R.R. & A.E. Rushing. 1990. Torus structure and development in the of Ulmus alata Michx., Celtis laevigata Wild., and Celtis occidentalis L. IAWA Bull. n.s. 11: 71–83. Dute, R.R., A.E. Rushing & J.W. Perry. 1990. Torus structure and development in species of Daphne. IAWA Bull. n.s. 11: 401–412. Evert, R.F. 2006. Esauʼs anatomy. John Wiley & Sons, Inc., New Jersey. Fengel, D. 1972. Structure and function of the membrane in softwood bordered pits. Holzfor- schung 26: 1–9. Fujikawa, S. & S. Ishida. 1972. Study on the pit of wood cells using scanning electron micros- copy. III. Structural variation of bordered pit membrane on the radial wall between tracheids in Pinaceae species. J. Jap. Wood Res. Soc. 18: 477–483. Hacke, U.G., J.S. Sperry & J. Pittermann. 2004. Analysis of circular bordered pit function. II. Gymnosperm tracheids with torus-margo pit membranes. Amer. J. Bot. 91: 386–400. Imamura, Y. & H. Harada. 1973. Electron microscopic study on the development of the bordered pit in coniferous tracheids. Wood Sci. Technol. 7: 189–205. Imamura, Y., H. Harada & H. Saiki. 1974. Embedding substances of pit membranes in softwood tracheids and their degradation by enzymes. Wood Sci. Technol. 8: 243–254. Karnovsky, M.J. 1965. A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J. Cell Biol. 27: 137A. Kollmann, R. & C. Glockmann. 1991. III. On the mechanism of secondary formation of plasmo- desmata at the graft interface. Protoplasma 165: 71–85. Kragler, F., W.J. Lucas & J. Monzer. 1998. Plasmodesmata: dynamics, domains and patterning. Ann. Bot. 81: 1–10. Krahmer, R.L. & W.A. Côté. 1963. Changes in coniferous wood cells associated with heartwood formation. Tappi 46: 42–49.

Downloaded from Brill.com10/06/2021 11:20:42PM via free access 288 IAWA Journal, Vol. 29 (3), 2008 Dute, Hagler & Black — Pit membranes of Abies and Metasequoia 289

Kwiatkowska, M. 1991. Autoradiographic studies on the role of plasmodesmata in the transport of gibberellin. Planta 183: 294–299. Liese, W. 1965. The structure of bordered pits in softwoods. In: W.A. Côté (ed.), Cellular ul- trastructure of woody : 271–290. Syracuse University Press, New York. McCann, M.C., B. Wells & K. Roberts. 1990. Direct visualization of cross-links in the primary plant . J. Cell Sci. 96: 323–334. Morrow, A.C. & R.R. Dute. 1999. Electron microscopic investigation of the coating found on torus-bearing pit membranes of Botrychium dissectum, the common grape . IAWA J. 20: 359–373. Murmanis, L. & I.B. Sachs. 1969. Seasonal development of secondary xylem in Pinus strobus L. Wood Sci. Technol. 3: 177–193. Nation, J.L. 1983. A new method using hexamethyldisilazane for preparation of soft insect tis- sues for scanning electron microscopy. Stain Technology 58: 347–351. Parham, R.A. & W.M. Baird. 1973. The bordered pit membrane in differentiating balsam fir. Wood & Fiber 5: 80–86. Pittermann, J., J.S. Sperry, U.G. Hacke, J.K. Wheeler & E.H. Sikkema. 2005. Torus-margo pits help conifers compete with angiosperms. Science 310: 1924. Sano, Y., Y. Kawakami & J. Ohtani. 1999. Variation in the structure of intertracheary pit mem- branes in Abies sachalinensis, as observed by field-emission scanning electron microscopy. IAWA J. 20: 375–388. Spurr, A.R. 1969. A low viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26: 31–45. Thomas, R. 1972. The ultrastructure of differentiating and mature bordered pit membranes from cypress (Taxodium distichum R. Rich.). Wood & Fiber 4: 87–94. Thomas, R.J. 1969. The ultrastructure of southern pine bordered pit membranes as revealed by specialized drying techniques. Wood & Fiber 1: 110–123. Timmell, T.E. 1979. Formation of compression wood in balsam fir (Abies balsamea). II. Ultra- structure of the differentiating xylem. Holzforschung 33: 181–191. Zimmermann, M.H. 1983. Xylem structure and the ascent of . Springer-Verlag, Berlin.

Downloaded from Brill.com10/06/2021 11:20:42PM via free access